Project Summary Despite strong anti-smoking efforts, at least 12% of American women cannot quit smoking when pregnant, resulting in more than 450,000 smoke-exposed infants born yearly. Maternal smoking during pregnancy adversely affects lung development as seen by lifelong decreases in pulmonary function and increased risk of asthma. In a randomized controlled trial (RCT), published in JAMA, we have provided evidence that maternal vitamin C supplementation (500 mg daily during pregnancy) can decrease the effects of maternal smoking during pregnancy on offspring lung function and the subsequent incidence of wheeze. The purpose of this ECHO application is to combine the offspring from the prenatal cohorts of two RCTs studying the effects of vitamin C supplementation to pregnant smokers on offspring respiratory outcomes and to determine if the innovative therapy of maternal vitamin C supplementation during pregnancy leads to prolonged improvements on offspring?s pulmonary function tests (PFTs) and respiratory health. To begin to understand mechanisms by which prenatal vitamin C may be preventing changes in the health of offspring of smokers, we examined gene specific DNA methylation from a subset of placentas, cord bloods and buccal swabs (ages 2-5) collected from subjects who were the offspring of prospectively-followed pregnant placebo- treated smokers, vitamin C-treated smokers, or non-smokers. In parallel with the effects of vitamin C on the reduction of the effects of maternal smoking on offspring PFTs and wheeze, the supplementary vitamin C prevented the majority of significant changes in DNA methylation induced by maternal smoking in both placentas and cord blood. Strikingly, this prevention of alterations in DNA methylation was still evident 2-5 years later in offspring buccal swabs. This preliminary data suggests that the mechanism underlying vitamin C?s prevention of some of the effects of maternal smoking on offspring pulmonary health involves the prevention of epigenetic changes induced by maternal smoking. Thus this application has 4 specific aims: 1, to create the combined cohort of offspring from the 2 RCTs of vitamin C supplementation to pregnant smokers; 2, to determine if the protective effects of maternal vitamin C supplementation to pregnant smokers on offspring pulmonary health extend to early adolescence; 3, to begin to characterize the epigenetic mechanisms that appear to underlie the effects of smoking during pregnancy on offspring lung health and on the protective effects of vitamin C; and 4, to gather core data for and interact with the overall ECHO study. Respiratory health will be assessed by longitudinal semi-annual, validated respiratory histories and yearly PFTs beginning at 3 years of age. Buccal swabs will be collected yearly and blood at 6, 9 and 12 years to provide DNA for genetic analysis. DNA methylation changes will be measured by methylation chips and bisulfite sequencing. This study will address how smoking during pregnancy affects respiratory outcomes, provide mechanistic and therapeutic insights, and provide subjects and data to ECHO to address other key outcomes.